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. 2014 May 20;111(20):7403-8.
doi: 10.1073/pnas.1402911111. Epub 2014 May 6.

Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex

Si Ming Man  1 , Lee J Hopkins  1 , Eileen Nugent  2 , Susan Cox  3 , Ivo M Glück  1 , Panagiotis Tourlomousis  1 , John A Wright  1 , Pietro Cicuta  2 , Tom P Monie  4 , Clare E Bryant  5
Affiliations

Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex

Si Ming Man et al. Proc Natl Acad Sci U S A. .

Abstract

Pathogen recognition by nucleotide-binding oligomerization domain-like receptor (NLR) results in the formation of a macromolecular protein complex (inflammasome) that drives protective inflammatory responses in the host. It is thought that the number of inflammasome complexes forming in a cell is determined by the number of NLRs being activated, with each NLR initiating its own inflammasome assembly independent of one another; however, we show here that the important foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) simultaneously activates at least two NLRs, whereas only a single inflammasome complex is formed in a macrophage. Both nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and nucleotide-binding domain and leucine-rich repeat pyrin domain 3 are simultaneously present in the same inflammasome, where both NLRs are required to drive IL-1β processing within the Salmonella-infected cell and to regulate the bacterial burden in mice. Superresolution imaging of Salmonella-infected macrophages revealed a macromolecular complex with an outer ring of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain and an inner ring of NLRs, with active caspase effectors containing the pro-IL-1β substrate localized internal to the ring structure. Our data reveal the spatial localization of different components of the inflammasome and how different members of the NLR family cooperate to drive robust IL-1β processing during Salmonella infection.

Keywords: ASC; bacteria; caspase-1; caspase-8; innate immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Spatial organization of the ASC speck. (A) Immunolabeling of endogenous ASC in primary BMMs infected with S. Typhimurium. (B) Endogenous ASC oligomerized in unprimed or LPS-primed BMMs following S. Typhimurium infection. (C) Confocal microscopy imaging of endogenous immunolabeled ASC and fluorescent-labeled inhibitor of caspases (FLICA) staining of caspase-1 in human THP-1 macrophages infected with S. Typhimurium. (D) Bayesian localization superresolution microscopy of endogenous ASC and caspase-1 in C. (E) Multiple cross-sections of the ASC–caspase-1 speck. Arrowheads indicate side filaments of ASC coming off the external region of the ring-like structure.
Fig. 2.
Fig. 2.
NLRs reside in the ASC speck following S. Typhimurium infection of macrophages. Bayesian localization superresolution microscopy of endogenous immunolabeled ASC and NLRP3 (A), endogenous immunolabeled NLRP3 and FLICA-stained caspase-1 (B), and endogenous immunolabeled NLRC4 and FLICA-stained caspase-1 in human THP-1 macrophages infected with S. Typhimurium (C) are shown.
Fig. 3.
Fig. 3.
Coordinated activation of NLRs is required for IL-1β production induced by S. Typhimurium infection. Unprimed WT, Nlrc4−/−, and Nlrp3−/− primary BMMs were infected with S. Typhimurium SL1344 (A) or its isogenic mutants ΔfliC, ΔfljB, ΔfliCΔfljB, ΔprgJ, and ΔfliCΔfljBΔprgJ (B) (MOI of 1) for the indicated times. (A and B) Supernatant was collected, and the levels of IL-1β were measured using ELISA. (C) Procaspase-1 (Pro–casp-1), cleaved caspase-1 (Casp-1) p10, pro–IL-1β, and cleaved IL-1β p17 were detected in the supernatant (SN) or cell lysate using Western blotting. β-actin was used as a loading control. Data shown are representatives of two (C) and three (A and B) experiments.
Fig. 4.
Fig. 4.
NLRC4 and NLRP3 have nonredundant roles in the host defense against sublethal murine S. Typhimurium infection. WT, Nlrc4−/−, Nlrp3−/−, and casp-1−/−(casp-11−/−) mice were infected i.v. with 1.33 × 104 cfu of S. Typhimurium strain M525P (a strain that establishes sublethal murine salmonellosis), and the mean bacterial load was determined in the spleen and liver after 1, 7, and 13 d of infection. Three to four animals of each genotype were used per group per time point. Data are from one set of experiments representative of two. ns, no statistical significance; **P < 0.01; ***P < 0.001.
Fig. 5.
Fig. 5.
NLRC4 and NLRP3 are recruited to the same Salmonella-induced ASC speck. (A) Unprimed WT primary BMMs were infected with S. Typhimurium expressing GFP for 2 or 24 h, and were stained for ASC and DNA. The number of ASC foci per infected BMM was counted. At least 180 infected BMMs (indicated by the presence of GFP-expressing S. Typhimurium) were counted in each of the four independent experiments. (B) Bayesian localization superresolution microscopy of endogenous NLRC4 and NLRP3 in THP-1 macrophages infected with S. Typhimurium. (C) Image analysis of the ring diameter of the NLRC4 and NLRP3 structures in B (n = 10).
Fig. 6.
Fig. 6.
Caspase-8 is recruited to the same Salmonella-induced ASC speck. (A and B) Bayesian localization superresolution microscopy of endogenous immunolabeled ASC and FLICA-stained caspase-8 in THP-1 macrophages infected with S. Typhimurium. (B) Multiple cross-sections of the ASC–Caspase-8 speck. Arrowheads indicate side filaments of ASC. Image analysis of the ring diameter of the ASC and caspase-8 structures (n = 7). (C) Schematic of ASC speck assembly in response to Salmonella infection in a macrophage.
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